The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention, and is not admitted to describe or constitute prior art to the present invention.
High-content screening (HCS) in cell-based systems uses living cells as tools in biological research to elucidate the workings of normal and diseased cells. HCS is also used to discover and optimizes new drug candidates.
High content screening is a combination of modern cell biology, with all its molecular tools, with automated high resolution microscopy and robotic handling. Cells are first exposed to chemicals or RNAi reagents. Changes in cell morphology are then detected using image analysis. Changes in the amounts of proteins synthesized by cells are measured using a variety of techniques such as the green fluorescent proteins fused to endogenous proteins, or by fluorescent antibodies.
At a cellular level, parallel acquisition of data on different cell properties, for example activity of signal transduction cascades and cytoskeleton integrity is the main advantage of this method in comparison to the faster but less detailed high throughput screening. While HCS is slower, the wealth of acquired data allows a more profound understanding of drug effects. In this sense, one of the goals of HCS in the acquisition of data in connection to the activity of signal transduction cascades is to determine the effect of different drugs in the signalling processes through the measurement of intracellular second messenger levels.
Second messengers are molecules that relay signals from receptors on the cell surface to target molecules inside the cell, in the cytoplasm or nucleus. They relay the signals of hormones like epinephrine (adrenaline), growth factors, and others, and cause some kind of change in the activity of the cell. They greatly amplify the strength of the signal. Secondary messengers are a component of signal transduction cascades. Among these second messengers, the cAMP and calcium provide the paradigm for the second messenger concept and are appreciated as ubiquitous and critical intracellular molecules that regulate many key processes in the cell.
Ideally, said measurement requires tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible that in turn are capable of monitoring the levels of second messengers in vivo by using a high content screening method.
For this, various fluorescent biosensors based on dynamically changing the fluorescent properties have been generated. In this sense, these types of biosensors are often based on a change in Fluorescent Resonance Energy Transfer (FRET). FRET is the process by which energy from an excited donor fluorophore is transferred to an acceptor fluorophore through radiationless dipole-dipole coupling. The efficiency of this energy transfer is highly dependent on the distance between (e.g. <10 nm for CFP/YFP) and the relative orientation of donor and acceptor fluorophore. However, FRET-based biosensors in the context of high content screening methods requires of a detection equipment of at least four filters, two for the excitation and two for the emission. In addition, due to the low intensity of the detection signal, the detection signal range and the screening sensibility are low. Lastly, the use of more than one fluorescence emission signal requires the use of more algorithms in order to correctly analyse the final signal.
Thus, there is still a need to develop improved methods or products for real time measurement of second messenger concentration within the dynamic environment of the living cell,